ORIGINAL ARTICLE
Circulation Journal Official Journal of the Japanese Circulation Society http://www. j-circ.or.jp
Arrhythmia/Electrophysiology
Reversal of Rivaroxaban-Induced Alterations on Hemostasis by Different Coagulation Factor Concentrates – In Vitro Studies With Steady and Circulating Human Blood – Gines Escolar, MD, PhD; Eduardo Arellano-Rodrigo, MD, PhD; Irene Lopez-Vilchez, PhD; Patricia Molina; Juan Sanchis, MD, PhD; Joan Carles Reverter, MD, PhD; Xavier Carne, MD, PhD; Joan Cid, MD, PhD; Jaume Villalta, MD, PhD; Dolors Tassies, MD, PhD; Ana M. Galan, PhD; Maribel Diaz-Ricart, PhD
Background: Despite the good safety of rivaroxaban, there is limited information on strategies for urgent reversal of its antihemostatic effects. Methods and Results: Alterations of hemostasis induced by rivaroxaban (230 ng/ml) were assessed by using several tests applied to steady and circulating human blood. Effects on thrombin generation (TG) and thromboelastometry (TEM) parameters were measured. Modifications in platelet adhesive, aggregating and procoagulant activities were evaluated in studies with circulating blood. The potential reversal of prothrombin complex concentrates (PCCs; 50 IU/kg), activated PCCs (aPCCs; 75 IU/kg), or recombinant factor VIIa (rFVIIa; 270 μg/kg) was evaluated. Impairment of TG parameters induced by rivaroxaban were corrected by the different concentrates (aPCC≥PCC>rFVIIa). Prolonged clotting times and reduced clot firmness caused by rivaroxaban on TEM tests were improved by different concentrates (rFVIIa≥aPCC>PCC). Rivaroxaban significantly reduced platelets and fibrin interactions with damaged vascular surfaces in perfusion studies. While alterations of platelet interactions were favourably counteracted by rFVIIa or aPCCs, reductions in fibrin formation were only partially restored by the different factor concentrates (rFVIIa>aPCC≥PCC). Conclusions: Rivaroxaban-induced alterations on coagulation parameters measured through assays performed under static conditions were easily reversed by the different concentrates. Studies under flow conditions revealed that these concentrates normalized the action of rivaroxaban on platelets, and significantly improved fibrin formation; although in the later case, levels were not restored to the pre-treatment value. (Circ J 2015; 79: 331 – 338) Key Words: Activated prothrombin complex concentrates; Prothrombin complex concentrates; Recombinant factor VIIa; Rivaroxaban
A
nticoagulation is essential for the prevention and treatment of different conditions, such as atrial fibrillation, deep vein thrombosis or pulmonary embolism. Rivaroxaban is a new oral anticoagulant with a selective inhibitory action on factor Xa.1 Different clinical trials have investigated the efficacy and safety of rivaroxaban compared with standard therapy in different clinical settings, and it is currently indicated for the prevention of stroke and systemic embolism in patients with non-valvular atrial fibrillation, prevention of venous thromboembolism in adult patients under-
going elective hip or knee replacement surgery, treatment of deep vein thrombosis and pulmonary embolism and the prevention of recurrent deep vein thrombosis and pulmonary embolism in adults.2–10 Despite the good safety profile of rivaroxaban shown in clinical trials,9,11,12 no definite information is available about possible strategies for the reversal of its antihemostatic effects in patients presenting with medical or surgical emergencies.13–16 The summary of the product characteristics for rivaroxaban recommends that in cases in which bleeding cannot be controlled, the use of PCC, aPCC or
Received August 18, 2014; revised manuscript received October 16, 2014; accepted November 9, 2014; released online December 8, 2014 Time for primary review: 41 days Department of Haemotherapy and Haemostasis, Hospital Clinic, Centre of Biomedical Diagnosis, Institute of Biomedical Research August Pi i Sunyer, University of Barcelona, Barcelona (G.E., E.A.-R., I.L.-V., P.M., J.C.R., J.C., D.T., A.M.G., M.D.-R.); Department of Cardiology, Hospital Clinico Universitario de Valencia, School of Medicine, University of Valencia, Valencia (J.S.); Department of Pharmacology, Hospital Clinic, Barcelona (X.C.); and Department of Internal Medicine, Hospital Clinic, Barcelona (J.V.), Spain Mailing address: Gines Escolar, MD, PhD, Department of Haemotherapy and Haemostasis, Hospital Clinic, 170 Villarroel Street, 08036 Barcelona, Spain. E-mail: [email protected] ISSN-1346-9843 doi: 10.1253/circj.CJ-14-0909 All rights are reserved to the Japanese Circulation Society. For permissions, please e-mail: [email protected] Circulation Journal Vol.79, February 2015
332
ESCOLAR G et al.
rFVIIa should be considered,17 although no clinical evidence is available to support such a recommendation.
Editorial p 289 Investigations of the subtle mechanisms involved in the antithrombotic action of rivaroxaban preventing formation of occlusive thrombi at the level of the damaged vasculature and reversal of its anticoagulant action are difficult to perform in patients that are receiving treatment while in clinical trials. The direct inhibitory action of rivaroxaban facilitates the development of experimental approaches in vitro to generate evidence on detailed mechanisms of action and possible reversal.18 In the present study, we evaluated the effects of the average Cmax concentration (230 ng/ml) achieved with a standard rivaroxaban dose of 20 mg/day, on platelet- and coagulationmediated mechanisms of hemostasis. We applied a series of laboratory tests performed on steady blood or plasma samples, and performed additional studies with circulating human blood. After establishing the effects of rivaroxaban in the different biomarkers of coagulation, we explored the effects of different coagulation factor concentrates, including prothrombin complex concentrates (PCCs), activated PCCs (aPCCs) or recombinant factor VIIa (rFVIIa), to reverse the alterations of hemostasis previously induced by rivaroxaban.
Methods Ethics Statement Our investigations were performed in accordance with the Directive 2010/63/EU of the European Parliament on the protection of animals used for scientific purposes, and the principles outlined in the Declaration of Helsinki. The study was approved by the Hospital Clinic Ethical Committee of Clinical Investigation. The protocol to isolate rabbit aortas to be used as thrombogenic substrata was also approved by the Animal Ethical Committee of the University of Barcelona. Study Material The study group consisted of 8 healthy volunteers who agreed to donate blood samples after written informed consent was obtained. Individuals who had received acetylsalicylic acid, non-steroidal anti-inflammatory or antiplatelet drugs within 7 days before blood sampling were excluded. Blood samples were collected into tubes (BD Vacutainer, Franklin Lakes, NJ, USA) containing citrate (final concentration of 13 mmol/L). Rivaroxaban was kindly provided by Bayer HealthCare. Experimental Design Rivaroxaban was initially dissolved in ethanol, and subsequently diluted in saline. Aliquots of rivaroxaban dilutions were added to blood samples to achieve a Cmax-equivalent plasma concentration of rivaroxaban after a steady 20 mg/day dose (230 ng/ml).19 Whole blood samples spiked with rivaroxaban were used to evaluate modifications in: (1) viscoelastic parameters of clot formation in whole blood using thromboelastometry (ROTEM, TEM International GmbH, Munchen, Germany); (2) dynamics of thrombin generation (TG) in plasma using the fluorogenic assay, Technothrombin TGA (Technoclone GmBH, Wien, Austria); and (3) perfusion studies with whole blood circulated through damaged vascular segments at a shear rate of 600/s, equivalent to that found in medium-sized cerebral arteries. Commercially available coagulation factor concentrates were
tested for determining their ability to reverse the antihemostatic actions of rivaroxaban at doses approved in the prescribing information for each concentrate: rFVIIa: Novoseven® 270 µg/kg (NovoNordisk, Bagsvaerd, Denmark); aPCC: Feiba® 75 U/kg (Baxter); and PCC: Beriplex® 50 IU/kg (CSL Behring GmbH, Marburg, Germany). Doses of these concentrates added to blood were calculated assuming a blood volume of 4,900 ml in an adult weighing 70 kg. Aliquots of the different concentrates were spiked in the blood samples to evaluate their potential corrective effect on the different laboratory tests. TG Assay (TGA) TG was evaluated in citrated platelet-poor plasma (PPP) samples. TG on citrated PPP was assessed with the fluorogenic assay, Technothrombin TGATM, by following the manufacturer’s instructions (Technoclone GmBH, Austria).20,21 The activation of the coagulation cascade was triggered by 2 different commercial reagents, Technothrombin® RC Low (RCL) containing a low concentration micelles of negatively charged phospholipids and recombinant human tissue factor, and Technothrombin® RD (RD) containing negatively charged phospholipids. Thromboelastometry Studies The dynamic thrombelastography of whole blood coagulation, using the ROTEM Analyser (PentapharmGmbH, Munchen, Germany) was investigated.22 For simplicity, the study focused on the analysis of the exTEM test (Rotem Thromboelastometry, Biometa, Spain) in TEM studies with citrated blood, recalcified with 6 mmol/L CaCl2. Three of them were assessed for the purpose of our studies: clotting time (CT), the time (s) elapsed from the measurement start until the amplitude of the forming clot reaches 2 mm; clot formation time (CFT), the time (s) from the start of clot formation until the tracing reaches 20 mm of amplitude; and maximum clot firmness (MCF), the maximum amplitude of the tracing reached (in mm).23,24 CT and CFT were indicators of the dynamics of clot formation. The MCF or clot amplitude gave information about clot strength and stability. ROTEM analyses were performed for a minimum of 45 min. Perfusion Studies Aliquots of blood were perfused through annular chambers exposing damaged vascular segments, as thrombogenic substrata. Aortas were extracted from young female New Zealand rabbits (2.8–3.0 kg) previously euthanized according to protocols approved by the Animal Ethical Committee of the University of Barcelona (number DAAM: 6632). Vessels were cleaned, everted, cut into segments and maintained in PBS.25 Perfusions studies were performed at a shear rate of 600/s for 10 min. Before it entered the flow chamber, citrate-anticoagulated blood was mixed with 6 mmol/L CaCl2. Perfused vessels were rinsed with PBS (0.15 mol/L), fixed with 2.5% glutaraldehyde (in 0.15 mol/L PBS) at 4°C for 24 h and processed histologically for further morphometric evaluation. Fibrin deposition and platelet interactions were evaluated as previously described.21 Statistical Analysis Data were expressed as mean ± standard error of the mean (SEM). The SPSS statistical package 17.0.0 (SPSS Inc, Chicago, IL, USA) was used for all analyses. Statistical analysis was performed with raw data using ANOVA. Comparative statistics were performed with respect to values in control studies
Circulation Journal Vol.79, February 2015
Hemostatic In Vitro Reversal of Rivaroxaban
333
Table 1. Effects of Rivaroxaban in Thrombin Generation Kinetics in Plasma Samples Triggered With Different Technothrombin® Reagents Lag phase (min)
Thrombin peak (nmol/L)
Time peak (min)
A) Phospholipids micelles and tissue factor (Technothrombin® RCL) 19.5±1.6
159.0±26.0
34.6±2.8
47.0±3.3**
30.5±6.5**
80.2±3.1**
RIV+rFVIIa
17.1±1.3††
103.1±13.4††
41.6±4.7††
RIV+aPCC
14.7±1.3*,††
138.0±20.5††
39.5±4.9††
RIV+PCC
34.3±9.6
73.9±24.5*
74.0±6.4*
Control RIV
B) Phospholipids micelles (Technothrombin® RD) Control
4.0±0.5
510.6±19.2
7.8±0.5
RIV
4.1±0.5
468.5±18.4*
11.3±0.9**
RIV+rFVIIa
3.7±0.3
445.9±15.7**
8.8±0.6*,††
RIV+aPCC
5.2±0.5*,†
931.6±33.2*,††
11.3±1.7*
RIV+PCC
4.6±0.5
847.7±93.3*,††
13.3±2.1*
RIV, Rivaroxaban 230 ng/ml; recombinant factor VIIa (rFVIIa), Novoseven® 270 μg/kg; activated prothrombin complex concentrates (aPCC), Feiba® 75 U/kg; prothrombin complex concentrates (PCC), Beriplex® 50 IU/kg. *P
Circulation Journal Official Journal of the Japanese Circulation Society http://www. j-circ.or.jp
Arrhythmia/Electrophysiology
Reversal of Rivaroxaban-Induced Alterations on Hemostasis by Different Coagulation Factor Concentrates – In Vitro Studies With Steady and Circulating Human Blood – Gines Escolar, MD, PhD; Eduardo Arellano-Rodrigo, MD, PhD; Irene Lopez-Vilchez, PhD; Patricia Molina; Juan Sanchis, MD, PhD; Joan Carles Reverter, MD, PhD; Xavier Carne, MD, PhD; Joan Cid, MD, PhD; Jaume Villalta, MD, PhD; Dolors Tassies, MD, PhD; Ana M. Galan, PhD; Maribel Diaz-Ricart, PhD
Background: Despite the good safety of rivaroxaban, there is limited information on strategies for urgent reversal of its antihemostatic effects. Methods and Results: Alterations of hemostasis induced by rivaroxaban (230 ng/ml) were assessed by using several tests applied to steady and circulating human blood. Effects on thrombin generation (TG) and thromboelastometry (TEM) parameters were measured. Modifications in platelet adhesive, aggregating and procoagulant activities were evaluated in studies with circulating blood. The potential reversal of prothrombin complex concentrates (PCCs; 50 IU/kg), activated PCCs (aPCCs; 75 IU/kg), or recombinant factor VIIa (rFVIIa; 270 μg/kg) was evaluated. Impairment of TG parameters induced by rivaroxaban were corrected by the different concentrates (aPCC≥PCC>rFVIIa). Prolonged clotting times and reduced clot firmness caused by rivaroxaban on TEM tests were improved by different concentrates (rFVIIa≥aPCC>PCC). Rivaroxaban significantly reduced platelets and fibrin interactions with damaged vascular surfaces in perfusion studies. While alterations of platelet interactions were favourably counteracted by rFVIIa or aPCCs, reductions in fibrin formation were only partially restored by the different factor concentrates (rFVIIa>aPCC≥PCC). Conclusions: Rivaroxaban-induced alterations on coagulation parameters measured through assays performed under static conditions were easily reversed by the different concentrates. Studies under flow conditions revealed that these concentrates normalized the action of rivaroxaban on platelets, and significantly improved fibrin formation; although in the later case, levels were not restored to the pre-treatment value. (Circ J 2015; 79: 331 – 338) Key Words: Activated prothrombin complex concentrates; Prothrombin complex concentrates; Recombinant factor VIIa; Rivaroxaban
A
nticoagulation is essential for the prevention and treatment of different conditions, such as atrial fibrillation, deep vein thrombosis or pulmonary embolism. Rivaroxaban is a new oral anticoagulant with a selective inhibitory action on factor Xa.1 Different clinical trials have investigated the efficacy and safety of rivaroxaban compared with standard therapy in different clinical settings, and it is currently indicated for the prevention of stroke and systemic embolism in patients with non-valvular atrial fibrillation, prevention of venous thromboembolism in adult patients under-
going elective hip or knee replacement surgery, treatment of deep vein thrombosis and pulmonary embolism and the prevention of recurrent deep vein thrombosis and pulmonary embolism in adults.2–10 Despite the good safety profile of rivaroxaban shown in clinical trials,9,11,12 no definite information is available about possible strategies for the reversal of its antihemostatic effects in patients presenting with medical or surgical emergencies.13–16 The summary of the product characteristics for rivaroxaban recommends that in cases in which bleeding cannot be controlled, the use of PCC, aPCC or
Received August 18, 2014; revised manuscript received October 16, 2014; accepted November 9, 2014; released online December 8, 2014 Time for primary review: 41 days Department of Haemotherapy and Haemostasis, Hospital Clinic, Centre of Biomedical Diagnosis, Institute of Biomedical Research August Pi i Sunyer, University of Barcelona, Barcelona (G.E., E.A.-R., I.L.-V., P.M., J.C.R., J.C., D.T., A.M.G., M.D.-R.); Department of Cardiology, Hospital Clinico Universitario de Valencia, School of Medicine, University of Valencia, Valencia (J.S.); Department of Pharmacology, Hospital Clinic, Barcelona (X.C.); and Department of Internal Medicine, Hospital Clinic, Barcelona (J.V.), Spain Mailing address: Gines Escolar, MD, PhD, Department of Haemotherapy and Haemostasis, Hospital Clinic, 170 Villarroel Street, 08036 Barcelona, Spain. E-mail: [email protected] ISSN-1346-9843 doi: 10.1253/circj.CJ-14-0909 All rights are reserved to the Japanese Circulation Society. For permissions, please e-mail: [email protected] Circulation Journal Vol.79, February 2015
332
ESCOLAR G et al.
rFVIIa should be considered,17 although no clinical evidence is available to support such a recommendation.
Editorial p 289 Investigations of the subtle mechanisms involved in the antithrombotic action of rivaroxaban preventing formation of occlusive thrombi at the level of the damaged vasculature and reversal of its anticoagulant action are difficult to perform in patients that are receiving treatment while in clinical trials. The direct inhibitory action of rivaroxaban facilitates the development of experimental approaches in vitro to generate evidence on detailed mechanisms of action and possible reversal.18 In the present study, we evaluated the effects of the average Cmax concentration (230 ng/ml) achieved with a standard rivaroxaban dose of 20 mg/day, on platelet- and coagulationmediated mechanisms of hemostasis. We applied a series of laboratory tests performed on steady blood or plasma samples, and performed additional studies with circulating human blood. After establishing the effects of rivaroxaban in the different biomarkers of coagulation, we explored the effects of different coagulation factor concentrates, including prothrombin complex concentrates (PCCs), activated PCCs (aPCCs) or recombinant factor VIIa (rFVIIa), to reverse the alterations of hemostasis previously induced by rivaroxaban.
Methods Ethics Statement Our investigations were performed in accordance with the Directive 2010/63/EU of the European Parliament on the protection of animals used for scientific purposes, and the principles outlined in the Declaration of Helsinki. The study was approved by the Hospital Clinic Ethical Committee of Clinical Investigation. The protocol to isolate rabbit aortas to be used as thrombogenic substrata was also approved by the Animal Ethical Committee of the University of Barcelona. Study Material The study group consisted of 8 healthy volunteers who agreed to donate blood samples after written informed consent was obtained. Individuals who had received acetylsalicylic acid, non-steroidal anti-inflammatory or antiplatelet drugs within 7 days before blood sampling were excluded. Blood samples were collected into tubes (BD Vacutainer, Franklin Lakes, NJ, USA) containing citrate (final concentration of 13 mmol/L). Rivaroxaban was kindly provided by Bayer HealthCare. Experimental Design Rivaroxaban was initially dissolved in ethanol, and subsequently diluted in saline. Aliquots of rivaroxaban dilutions were added to blood samples to achieve a Cmax-equivalent plasma concentration of rivaroxaban after a steady 20 mg/day dose (230 ng/ml).19 Whole blood samples spiked with rivaroxaban were used to evaluate modifications in: (1) viscoelastic parameters of clot formation in whole blood using thromboelastometry (ROTEM, TEM International GmbH, Munchen, Germany); (2) dynamics of thrombin generation (TG) in plasma using the fluorogenic assay, Technothrombin TGA (Technoclone GmBH, Wien, Austria); and (3) perfusion studies with whole blood circulated through damaged vascular segments at a shear rate of 600/s, equivalent to that found in medium-sized cerebral arteries. Commercially available coagulation factor concentrates were
tested for determining their ability to reverse the antihemostatic actions of rivaroxaban at doses approved in the prescribing information for each concentrate: rFVIIa: Novoseven® 270 µg/kg (NovoNordisk, Bagsvaerd, Denmark); aPCC: Feiba® 75 U/kg (Baxter); and PCC: Beriplex® 50 IU/kg (CSL Behring GmbH, Marburg, Germany). Doses of these concentrates added to blood were calculated assuming a blood volume of 4,900 ml in an adult weighing 70 kg. Aliquots of the different concentrates were spiked in the blood samples to evaluate their potential corrective effect on the different laboratory tests. TG Assay (TGA) TG was evaluated in citrated platelet-poor plasma (PPP) samples. TG on citrated PPP was assessed with the fluorogenic assay, Technothrombin TGATM, by following the manufacturer’s instructions (Technoclone GmBH, Austria).20,21 The activation of the coagulation cascade was triggered by 2 different commercial reagents, Technothrombin® RC Low (RCL) containing a low concentration micelles of negatively charged phospholipids and recombinant human tissue factor, and Technothrombin® RD (RD) containing negatively charged phospholipids. Thromboelastometry Studies The dynamic thrombelastography of whole blood coagulation, using the ROTEM Analyser (PentapharmGmbH, Munchen, Germany) was investigated.22 For simplicity, the study focused on the analysis of the exTEM test (Rotem Thromboelastometry, Biometa, Spain) in TEM studies with citrated blood, recalcified with 6 mmol/L CaCl2. Three of them were assessed for the purpose of our studies: clotting time (CT), the time (s) elapsed from the measurement start until the amplitude of the forming clot reaches 2 mm; clot formation time (CFT), the time (s) from the start of clot formation until the tracing reaches 20 mm of amplitude; and maximum clot firmness (MCF), the maximum amplitude of the tracing reached (in mm).23,24 CT and CFT were indicators of the dynamics of clot formation. The MCF or clot amplitude gave information about clot strength and stability. ROTEM analyses were performed for a minimum of 45 min. Perfusion Studies Aliquots of blood were perfused through annular chambers exposing damaged vascular segments, as thrombogenic substrata. Aortas were extracted from young female New Zealand rabbits (2.8–3.0 kg) previously euthanized according to protocols approved by the Animal Ethical Committee of the University of Barcelona (number DAAM: 6632). Vessels were cleaned, everted, cut into segments and maintained in PBS.25 Perfusions studies were performed at a shear rate of 600/s for 10 min. Before it entered the flow chamber, citrate-anticoagulated blood was mixed with 6 mmol/L CaCl2. Perfused vessels were rinsed with PBS (0.15 mol/L), fixed with 2.5% glutaraldehyde (in 0.15 mol/L PBS) at 4°C for 24 h and processed histologically for further morphometric evaluation. Fibrin deposition and platelet interactions were evaluated as previously described.21 Statistical Analysis Data were expressed as mean ± standard error of the mean (SEM). The SPSS statistical package 17.0.0 (SPSS Inc, Chicago, IL, USA) was used for all analyses. Statistical analysis was performed with raw data using ANOVA. Comparative statistics were performed with respect to values in control studies
Circulation Journal Vol.79, February 2015
Hemostatic In Vitro Reversal of Rivaroxaban
333
Table 1. Effects of Rivaroxaban in Thrombin Generation Kinetics in Plasma Samples Triggered With Different Technothrombin® Reagents Lag phase (min)
Thrombin peak (nmol/L)
Time peak (min)
A) Phospholipids micelles and tissue factor (Technothrombin® RCL) 19.5±1.6
159.0±26.0
34.6±2.8
47.0±3.3**
30.5±6.5**
80.2±3.1**
RIV+rFVIIa
17.1±1.3††
103.1±13.4††
41.6±4.7††
RIV+aPCC
14.7±1.3*,††
138.0±20.5††
39.5±4.9††
RIV+PCC
34.3±9.6
73.9±24.5*
74.0±6.4*
Control RIV
B) Phospholipids micelles (Technothrombin® RD) Control
4.0±0.5
510.6±19.2
7.8±0.5
RIV
4.1±0.5
468.5±18.4*
11.3±0.9**
RIV+rFVIIa
3.7±0.3
445.9±15.7**
8.8±0.6*,††
RIV+aPCC
5.2±0.5*,†
931.6±33.2*,††
11.3±1.7*
RIV+PCC
4.6±0.5
847.7±93.3*,††
13.3±2.1*
RIV, Rivaroxaban 230 ng/ml; recombinant factor VIIa (rFVIIa), Novoseven® 270 μg/kg; activated prothrombin complex concentrates (aPCC), Feiba® 75 U/kg; prothrombin complex concentrates (PCC), Beriplex® 50 IU/kg. *P
